Figure 6.
CBP-mediated acetylation facilitates FAM134B phosphorylation by CAMKII to further boost ER-phagy. (A) Mimicking permanent acetylation by K160Q enhanced S151 phosphorylation. HEK293T cells transfected with FAM134B (WT)-Flag, FAM134B (K160R)-Flag and FAM134B (K160Q)-Flag were treated with Tg (1 μM) for 0, 0.5, 1 h. IP was performed with anti-Flag beads, which was followed by Western blot (WB) for S151 phosphorylation. (B–E) CBP inhibition by small compounds or shRNA KD significantly reduced FAM134B phosphorylation at S151. Cells were treated with Tg (1 μM) for 1 h or C646 (10 μM) for 3 h. Cells were collected and analyzed for proteins as indicated by Western blot. Quantification of K160 acetylation and S151 phosphorylation is shown in C and E (n = 3 experimental replicates). Data are shown as means ± SEM and analyzed with one-way ANOVA. **P < 0.01, ***P < 0.001. (F) CBP WT instead of catalytic activity-dead mutant rescued FAM134B K160 acetylation in CBP KD cells. The indicated proteins were detected by Western blot. (G) Mimicking permanent acetylation by K160Q facilitated the interaction with CAMK2B. FAM134B (WT)-Flag, FAM134B (K160R)-Flag, and FAM134B (K160Q)-Flag were expressed individually in HEK293T cells, which simultaneously expressed HA-CAMK2B. IP was performed with anti-HA beads, which was followed by Western blot for FAM134B-Flag. The experiments were performed twice. (H) FAM134B K160 acetylation promotes S151 phosphorylation. Purified recombinant FAM134B proteins were pulled down by HA-CAMK2B which purified from HEK293T cells. In vitro kinase assay was performed. The mixture was incubated with 400 µM ATP and 1× CAMK2B kinase buffer at 30°C for 10 min. The indicated proteins were detected by Western blot. (I) Dynamic K160 acetylation and S151 phosphorylation of FAM134B. HEK293T cells were treated with Tg (1 μM) for different time points as indicated and BafA1 (10 nM) to block degradation. IP was performed with an antibody to acetylated lysine. The indicated proteins were detected by Western blot. (J) Measurement of interaction between HA-CBP and FAM134B-Flag by Western blot in HEK293T cells treated with 1 μM of Tg for different time points or DMSO as indicated in the presence of BafA1 (10 nM). The experiments were performed twice. (K) Measurement of interaction between SIRT7-Myc and FAM134B-Flag by Western blot in HEK293T cells treated with 1 μM of Tg for different time points or DMSO as indicated in the presence of BafA1 (10 nM). The experiments were performed twice. (L) Schematic representation of dynamic acetylation and phosphorylation of FAM134B to cope with ER stress. Molecular weight measurements are in kD. Source data are available for this figure: SourceData F6.

CBP-mediated acetylation facilitates FAM134B phosphorylation by CAMKII to further boost ER-phagy. (A) Mimicking permanent acetylation by K160Q enhanced S151 phosphorylation. HEK293T cells transfected with FAM134B (WT)-Flag, FAM134B (K160R)-Flag and FAM134B (K160Q)-Flag were treated with Tg (1 μM) for 0, 0.5, 1 h. IP was performed with anti-Flag beads, which was followed by Western blot (WB) for S151 phosphorylation. (B–E) CBP inhibition by small compounds or shRNA KD significantly reduced FAM134B phosphorylation at S151. Cells were treated with Tg (1 μM) for 1 h or C646 (10 μM) for 3 h. Cells were collected and analyzed for proteins as indicated by Western blot. Quantification of K160 acetylation and S151 phosphorylation is shown in C and E (n = 3 experimental replicates). Data are shown as means ± SEM and analyzed with one-way ANOVA. **P < 0.01, ***P < 0.001. (F) CBP WT instead of catalytic activity-dead mutant rescued FAM134B K160 acetylation in CBP KD cells. The indicated proteins were detected by Western blot. (G) Mimicking permanent acetylation by K160Q facilitated the interaction with CAMK2B. FAM134B (WT)-Flag, FAM134B (K160R)-Flag, and FAM134B (K160Q)-Flag were expressed individually in HEK293T cells, which simultaneously expressed HA-CAMK2B. IP was performed with anti-HA beads, which was followed by Western blot for FAM134B-Flag. The experiments were performed twice. (H) FAM134B K160 acetylation promotes S151 phosphorylation. Purified recombinant FAM134B proteins were pulled down by HA-CAMK2B which purified from HEK293T cells. In vitro kinase assay was performed. The mixture was incubated with 400 µM ATP and 1× CAMK2B kinase buffer at 30°C for 10 min. The indicated proteins were detected by Western blot. (I) Dynamic K160 acetylation and S151 phosphorylation of FAM134B. HEK293T cells were treated with Tg (1 μM) for different time points as indicated and BafA1 (10 nM) to block degradation. IP was performed with an antibody to acetylated lysine. The indicated proteins were detected by Western blot. (J) Measurement of interaction between HA-CBP and FAM134B-Flag by Western blot in HEK293T cells treated with 1 μM of Tg for different time points or DMSO as indicated in the presence of BafA1 (10 nM). The experiments were performed twice. (K) Measurement of interaction between SIRT7-Myc and FAM134B-Flag by Western blot in HEK293T cells treated with 1 μM of Tg for different time points or DMSO as indicated in the presence of BafA1 (10 nM). The experiments were performed twice. (L) Schematic representation of dynamic acetylation and phosphorylation of FAM134B to cope with ER stress. Molecular weight measurements are in kD. Source data are available for this figure: SourceData F6.

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